A subset of T cells, termed Th2 cells, is required for the generation of allergic immune responses. Th2 cells express three signature cytokines, IL-4, IL-5, and IL-13, each of which makes a specific contribution to allergic pathology. IL-4 drives Th2 differentiation and plays a dominant role in antibody class switching to IgE, IL-5 is the major eosinophil active cytokine, and IL-13 drives eosinophil-rich tissue inflammation though its upregulation of adhesions molecules and chemokines. We hypothesized heterogeneity within the Th2 lineage would yield Th2 subpopulations with different cytokine expression, effector functions and roles in disease pathogenesis. Using polychromatic flow cytometry, we found two dominant subpopulations within the human CD4 Th2 compartment: IL-5- Th2 cells, which co-expressed IL-4 and IL-13, and IL-5+ Th2 cells, which expressed all three Th2 cytokines. These two populations of Th2 cells could be demonstrated directly ex vivo as well as in T cell clones and short-term lines. Notably, only 15% of the Th2 cells isolated directly ex vivo from non-allergic subjects were IL-5+ Th2 cells. IL-5+ Th2 cells exhibited phenotypic markers of highly differentiated memory T cells, suggesting that the IL-5+ and IL-5- Th2 cells are more and less highly differentiated Th2 cells, respectively. We further tested this hypothesis by examining Th2 cytokine coexpression during serial rounds of in vitro Th2 differentiation. By the second round, two-thirds of the cells expressed IL-4 or IL-13, but <10% of the cells expressed IL-5. In each of the 5 rounds analyzed, there was a consistent and markedly delayed acquisition of the IL-5+ relative to the IL-5- Th2 phenotype. Microarray transcription profiling of the two Th2 subpopulations revealed approximately 150 differentially expressed genes between the IL-5- and IL5+ Th2 subpopulations. In summary, these findings demonstrate that Th2 cells consist of two subpopulations: IL-5+ and IL-5- Th2 cells, and that IL-5+ Th2 cells represent a phenotypically distinct subpopulation of highly differentiated Th2 cells. These results suggest that IL-5+ Th2 cells have different functional properties than IL-5- Th2 cells and may make a unique contribution to allergic inflammation. Understanding IL-5 gene regulation is important in developing effective strategies targeting eosinophilic inflammation.